Death from opioid overdose is primarily due to respiratory depression, yet little is known of the cellular mechanisms of opioids on respiratory-controlling neurons. Breathing is controlled by a highly interconnected network of neurons in the brainstem. Opioid-induced respiratory depression is due to activation of mu opioid receptors located throughout in the brainstem respiratory network, including the Klliker-Fuse (KF) in the pons and respiratory control areas in the medulla. There are conflicting results regarding the importance of these areas in opioid-induced respiratory depression. Based on preliminary data, we propose that opioids act on the respiratory network collectively to cause respiratory depression rather than one area in isolation. There are major voids in the understanding of opioid modulation of the respiratory circuitry, especially inhibition of transmitter release. The goal of this proposal is to determine opioid regulation of identified projections between respiratory controlling brainstem neurons in adult mice with fully developed respiratory circuitry. It will focus on three brainstem areas that express mu opioid receptors and have dense reciprocal projections: the preBtzinger complex and Btzinger complex in the medulla and the KF in the pons. The hypothesis is that presynaptic opioid receptors regulate synaptic connections between these areas to mediate opioid-induced respiratory depression. A combination of approaches will be used to test this hypothesis on the cellular, circuit and behavioral level. In Aim 1, opioid regulation of excitatory projections from the KF to the medulla will be defined using brain slice electrophysiology and optogenetics. Then, in vivo experiments will test the role of projections to the medulla in mediating respiratory depression caused by systemic opioid administration in awake adult animals. In Aim 2, the opioid sensitivity of synapses from medullary projection neurons onto KF neurons will be determined using brain slice electrophysiology and optogenetics. A unique arterially perfused preparation that maintains an intact brainstem and ?in vivo-like? respiratory cycle will be used to determine if medullary projection neurons control the activity of single KF neurons. In Aim 3, the vulnerability of presynaptic mu opioid receptors in the KF to chronic opioid treatment will be determined using brain slice electrophysiology. Results from this project will provide mechanistic detail on opioid control of the pontomedullary respiratory circuitry that leads to respiratory depression. This may help identify strategies to counter opioid-induced respiratory depression and also inform on how synaptic mechanisms affect behavior.